Sequencing in a Flash

The 454 method avoids several of the more time-­consuming steps of conventional sequencing, such as the separation of strands by size. Unlike Sanger sequencing, it doesn’t terminate chains: it records bases as they’re added to a growing strand. First, a DNA molecule is randomly chopped into different lengths. Then each fragment is stripped into single strands, and each strand is attached to a separate tiny bead. A biochemical process copies the single strands, so that 10 million clones jut out from each bead. Each bead is then packed into one of the 1.6 million wells. As, Cs, Ts, and Gs wash over the wells sequentially to synthesize new complementary strands.

Here’s the truly clever part: using a method first described by Pål Nyrén and coworkers at Sweden’s Royal Institute of Technology, 454’s sequencer instantly records when a base is added to each strand by exploiting the fact that the binding reaction releases a chemical called a pyrophosphate. In the wells of the 454 machine, the pyrophosphate is captured by a chemical cascade that ends up flicking on the enzyme luciferase (which occurs naturally in fireflies)–emitting a burst of light. A standard charge-­coupled device of the kind used in digital cameras and telescopes detects each flash, reading off the sequence of As, Cs, Ts, and Gs in each fragment. The process can read about 200 to 300 bases in a row. As in conventional sequencing, computers then look for matching sequences at the end of one fragment and the start of another, piecing the fragments back together in the correct order.

The sequencer that 454 brought to market in October 2005 had a few serious limitations. It could read only 100 bases in a row (the longer the stretch of bases in each sequenced fragment, the easier it is to assemble a complete genome), and it also had trouble accurately mapping repetitive stretches–say, six As back to back. But Rothberg says 454’s philosophy was “Get it out early; get it accepted.” The company first targeted “early adopters” like Broad’s Lander, hoping they would soon publish findings that relied on the sequencer. “You’ve got to get early guys first, but the rest of the guys, the followers, are where the market is,” says ­Rothberg. “And they read peer-reviewed papers.”

NeanderthalsOne paper by an early adopter that received widespread attention from scientists and the public alike was a study of Neanderthal DNA led by Svante Pääbo of the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany. Neanderthals, the closest species to modern humans, disappeared some 30,000 years ago, and more speculation than fact surrounds their genetic relationship to us. Though Pääbo had done some previous studies with Neanderthal DNA, anything beyond rudimentary analysis had proved too difficult and costly. The problem is that over thousands of years, the few known samples of Neanderthal DNA from fossils have been degraded to short fragments of around 50 to 75 base pairs. In addition, the DNA is often contaminated with genetic material from microörganisms and the modern humans who have handled the fossils. But ­Rothberg believed that the 454 machine could analyze many short sequences at little cost, generating enough information to let scientists sift ancient treasures from junk. Rothberg cold-called Pääbo, who agreed to collaborate.